Proteins having a variety of functions are widely utilized for commercial uses such as pharmaceutical products and industrial enzymes, and also for research uses in which proteins are used to elucidate various biological phenomena. Thus, proteins are substances essential in improvements in quality of life and scientific advancements. As means for producing these various proteins in large quantities at a low cost with a high reproducibility, techniques for producing proteins using recombinant organisms and techniques for purifying the proteins have been developed.
The techniques for producing proteins using recombinant organisms utilize animal cells (Non-Patent Literature 1) and microorganisms (Non-Patent Literature 1). The animal cells used include CHO cells (Non-Patent Literature 2), while the microorganisms used include Escherichia coli, yeasts (Non-Patent Literature 3), and the like. For example, there is a protein secretory production system using Corynebacterium glutamicum (hereinafter may be abbreviated as C. glutamicum) as the microorganism (Patent Literature 1).
Main methods for purifying proteins produced by recombinant organisms include a method utilizing properties of a protein itself, and a method by adding a sequence used for purification to a protein and utilizing properties of the added sequence.
The method utilizing properties of a protein itself includes chromatography and liquid-solid separation.
The chromatography uses chromatographic matrixes having various properties. The chromatography utilizes an interaction between a protein and a chromatographic matrix, or the molecular sieving effect of the chromatographic matrix (Non-Patent Literature 4). The interaction between a protein and a chromatographic matrix includes electrostatic interaction, hydrogen bond, hydrophobic interaction, specific interaction, and the like (Non-Patent Literatures 4 and 5).
The liquid-solid separation is a separation method including: insolubilizing (i.e., making solid) a protein in a solubilized state by changing the solution conditions, obtaining a solid component by a simple process such as centrifugation, and bringing the separated solid component into a solubilized state again. Specific examples of the means for insolubilizing the protein include isoelectric point precipitation (Non-Patent Literature 6), salting out (Non-Patent Literature 7), precipitation using an organic solvent (Non-Patent Literature 8), precipitation using a water-soluble polymer (Non-Patent Literature 9), and the like.
The isoelectric point precipitation utilizes a property in which the solubility of a protein becomes lowest at an isoelectric point thereof. The salting out, the precipitation using an organic solvent, and the precipitation using a water-soluble polymer utilize a property in which the solubility of a protein is decreased in the presence of the salt, the organic solvent, or the water-soluble polymer, each of which is at a high concentration. Another insolubilizing means is protein aggregation (Non-Patent Literatures 10 and 11). The protein aggregation may be particularly effective means when it is possible to select conditions for aggregating a protein other than a protein to be purified while leaving the protein to be purified in a solubilized state.
The method by adding a sequence used for purification to a protein and utilizing properties of the added sequence includes a method utilizing properties of the added sequence itself, and a method utilizing an interaction between the added sequence and a substance other than the added sequence.
The method utilizing properties of the added sequence itself includes a method utilizing elastin, which undergoes a phase transition to become insoluble with the temperature change (Non-Patent Literature 12), a method utilizing MISTIC, which forms a soluble assembly with the pH change (Patent Literature 2), and the like.
In the method utilizing an interaction between the added sequence and a substance other than the added sequence, the substance other than the added sequence is often disposed on a chromatographic matrix (Non-Patent Literature 13). The interaction between the added sequence and the substance other than the added sequence includes electrostatic interaction, hydrogen bond, hydrophobic interaction, specific interaction, and the like.